Transport across membranes and the influence of molecules associated with a membrane such as membrane proteins are important to study for instance when developing new drugs and also in many other contexts.
In 1895 Overton suggested that molecules permeate the cells in the same relative order as their oil-water partition coefficient [1] and in 1943 Danielli proposed that a continuous lipid bilayer acts as a diffusion barrier determining the rate of passive diffusion across cellular membranes [2]. However, the first means to directly study permeation across an isolated lipid bilayer became available in the early 60-ies through the development of means to prepare single lipid bilayers separating two aqueous compartments [3]. While this technique is well suited for permeability studies of charged solutes, the standard approach of today for studies of passive and active transport of non-electrolytes, including water transport and drug uptake, relies on measurements of osmotic-induced size changes of suspended lipid vesicles (so called liposomes) [4]. This method is based on dynamic light scattering (DLS) measurements of variations in scattered light intensity as the liposome dimension changes in response to osmotically induced water transfer across the lipid membrane, upon which the liposomes first (<1 ms) shrink, as water diffuse out of the liposomes, and subsequently swell as water reenters the liposomes driven by the inward permeation of solute molecules [5]. Although successfully applied in numerous studies on the nature of passive and active solute permeation [6], the method is restricted by the fact that a change in liposome size is an indirect effect, which does not necessarily correlate with the actual solute transport. In addition, since not only the liposome size, but also liposome motion, solute refractive index, and membrane aggregation contribute to the intensity of the scattered light, the quantification of solute transfer is not always straightforward [7,8]. From a practical perspective, this methods also suffers from low signal-to-noise ratios, which means that averaging from multiple data series is generally required to resolve kinetic traces. Furthermore, since measurements are performed on suspended liposomes, the method is not compatible with parallel or sequential screening of the very same liposome sample. This, in turn, means that substantial amounts of material are generally needed. Besides somewhat improved sensitivities, these limitations holds also for a less wide-spread method, in which osmotically induced liposome size fluctuations are recorded by monitoring changes in self-quenching of liposome-entrapped fluorophores upon liposome shrinkage and concomitant increase in fluorophore concentration [9].
The possibility to screen multiple recognition events either sequentially or simultaneously is one of the main advantages of surface-based bioanalytical sensor technologies, where the very same set of surface-immobilized probe molecules is exposed to a series of different compounds in an automated fashion. An additional reason for the emerging importance of these sensors in life-science stems from the fact that they can be combined with micro-fluidic handling [10], which makes them compatible with small sample volumes and thus well suited for measurements on rare and non-abundant substances. Surface plasmon resonance (SPR) is today the dominating surface-based bioanalytical sensor. It is based on excitation of laterally propagating surface plasmons at planar metal (usually gold) substrates, where the condition for SPR excitation is extremely sensitive to changes in interfacial refractive index, Δninterface, induced by for example biomolecular binding within a region in close proximity to the surface (typically hundreds of nanometers). Hence, by immobilizing probe molecules on the surface, binding of targets to the immobilized probes can be monitored in real time via a response which to a good approximation is proportional to Δninterface [11,12].
Other known devices for studying transport across membranes include liposomes in a solution. To the liposome membrane there are associated for instance a membrane protein of interest and the membrane protein mediated transport of an agent across the membrane is studied using methods involving for instance fluorescence and/or radioactivity measurements.
US 2004/0033624 disclose a membrane receptor reagent and assay device. Liposomes are tethered to a surface with anchor groups. The surface comprises reagent ligands that are tethered to the surface. The ligands are able to bind reversibly to a receptor in the liposome membrane. The membrane protein in the liposome is associated with a molecule with the capability to be excited by emitted energy from the surface and thereby produces a detectable signal. The binding of the membrane protein in the liposome membrane to the reagent ligands on the surface tend to pull the liposomes towards the surface. A test molecule with affinity to the membrane protein will bind competitively to the membrane protein and to some extent replace the reagent ligand on the surface. This will result in that membrane proteins come off the surface and are redistributed in the liposome membrane, while the liposome still is anchored to the surface. The membrane proteins will have a larger average distance from the surface and thereby they receive less energy from the surface, since the energy transfer from the surface is distance dependent. This can be detected. The method in US 2004/0033624 requires some kind of label such as a fluorophore in order to function.
In the assays for the measurement of membrane protein mediated transport across a membrane according to the state of the art, there is room for an improvement regarding the amount of membrane protein that has to be used. Membrane proteins are often difficult and expensive to purify in substantial quantities.
In the state of the art there is also room for improvement because a label has to be used in many assays. Labelling is not always possible since it may alter the structure and function of the analyte/receptor and interfere with the molecular interaction that is to be investigated. In addition fluorescent markers are hydrophobic, which can cause unspecific background binding.